Poly-γ-glutamic Acid (γ-pga) & Pudding Health Kit

Transcription

1 Nankai igem 2015 Poly-γ-glutamic Acid (γ-pga) & Pudding Health Kit

2 igem Nankai Team Nankai family 2015 Puding Health Kit 2015

3 Background Poly-γ-glutamic Acid (γ-pga) & Pudding Health Kit

4 Poly-γ-glutamic Acid (γ-pga) Unique Qualities Naturally occurring polyamide consisting of D/L-glutamate monomers. The amide linkages in γ-pga are formed between the α-amino group and the γ-carboxyl group The Molecular Weight is about 400,000

5 Poly-γ-glutamic Acid (γ-pga) Nontoxicity Biodegradability Water-solubility 4 5 Abundant carboxyl groups 6 Water absorption Potential modification

6 Applications of γ-pga Food Agriculture Biodegradable material Medicine Skin care products

7 Pudding Health Kit Wound Repairing Medicine γ-pga based hydrogel Pudding Health Kit Pudding Health Kit is a γ-pga based hydrogel containing wound repairing medicines.

8 Pudding Care Rescue Healing wounds

9 Production of γ-pga Glutamate-dependent Strains Glutamate-independent strains High fermentation cost Complex fermentation process High productivity Low fermentation cost Simple fermentation process Low productivity

10 Bacillus amyloliquefaciens strain Glutamate-independent strain isolated from fermented food. A B Can produce γ-pga with sucrose as its carbon source and ammonium sulfate as its nitrogen source. The construction of a glutamate-independent strain with high γ-pga yield and biomedical applications.

11 Experiment Poly-γ-glutamic Acid (γ-pga) & Pudding Health Kit

12 Metabolic flux of γ-pga synthesis γ-pga Sucrose Pyruvic acid 2- oxoglutarate pgsbca odha, odhb Glutamate Acetyl-CoA Succinyl-CoA

13 Part 1 Metabolic toggle switch

14 Metabolic toggle switch construction IPTG P laci laci P grac xylr P xyl odhab

15 Metabolic toggle switch verification IPTG P laci laci xylr bgab P grac P xyl P laci P grax xylr Terminator P xyl LacI Er R pht01-xylr ORF ori1030 ori1030 pcb 9045bp pcb-p xyl bgab Ap R Er R oriec oriec Ap R

16 Metabolic toggle switch verification Plasmids digested by double endonucleases. Marker III (M), control plasmids (1), digested plasmids(2).

17 Metabolic toggle switch function assay

18 Metabolic toggle switch construction IPTG P laci laci xylr odhab P grac P xyl P laci P grax xylr LacI Er R pht01-xylr ORF Ap R oriec

19 Metabolic toggle switch construction (A)pKSU-P xyl plasmid verification. (M)Marker III, (1)complete plasmids, (2) digested plasmids.(b) Confirmation of the B. amyloliquefaciens NK-TP. (M) Maker III, (1, 2) NK- TP strain, (3) NK-1 control strain.

20 Production increase in toggle off mode 63% 88% IPTG adding time

21 Part 2 Promoters replacement

22 Genes encoding γ-pga synthase There are three genes encoding protein to catalyze synthesis of γ-pga. There are three genes encoding protein to catalyze synthesis of γ-pga.

23 Promoter replacement P amya P 43 A 2up P bca pgsb pgsc pgsa BJ 27up C 2up

24 Promoter construct Plasmids verification. Marker III (M), control plasmids(1), digested plasmids(2)

25 Promoter strength assay Promoter strength assay in B.amyloliquefaciens NK-1

26 Promoter replacement Plasmids verification. Marker III (M), control plasmids (1), digested plasmids(2).

27 Promoter replacement Confirmation of the B. amyloliquefaciens NK-BJ27up. Marker III (M), NK-BJ27up (1,2,3), NK-1(4).

28 Parts submission

29 Application Poly-γ-glutamic Acid (γ-pga) & Pudding Health Kit

30 Chronic wound recovering process Day 7 Day 14 Day 21 H & E staining Masson staining Prolonged inflammation Reactive radicals Infections & fester H & E (A) and Masson (B) staining of a recovering chronic wound

31 SOD-PGA hydrogel wound dressing Keep moist Neutralize radicals Block out bacteria

32 PNIPAM/γ-PGA hydrogels

33 Temperature-sensitive PNIPAM/γ-PGA hydrogels Room temperature Above 32

34 SOD Release of PNIPAM/γ-PGA hydrogels

35 Wound healing effect Control group (3M wound dressing & gauze) PNIPAN/γ-PGA hydrogel SOD-PNIPAN/ γ-pga hydrogel

36 Wound healing effect Day 7 Day 14 Day 21 Control group SOD-PNIPAN/ γ-pga hydrogel H & E staining of a diabetic wound in recovery

37 Wound healing effect Day 7 Day 14 Day 21 Control group SOD-PNIPAN/ γ-pga hydrogel Masson staining of a diabetic wound in recovery

38 Human Practice Public Engagement & ishare Information Sharing Platform

39 Content in this Presentation Communication &Public Engagement ishare -Information Sharing Platform

40 Communication & Public Engagement

41 Nankai Univeristy& Tianjian University Communication and Win-win Collaboration N a n k a i & T i a n j i n i G E M Te a m i n i G E M M e e t u p P e k i n g C C i C

42 Nankai &ZJU &SYSU University In the near future, we will further communicate with more igem teams and build up collaborations. We have the same wish for a better team! Let s communicate and collaborate!

43 T h e 3 rd S Y M P O SIUM O F B O H AI - R I M M I C R O B I O L O GY

44 igem Meetup Peking

45 T h e 3 rd S Y M P O S I U M O F B O H A I - R I M M I C R O B I O L O GY igem Meetup Peking Not only can we get precious ideas from experts and other teams but also broaden our horizons on synthetic biology

46 ishare -Information Sharing Platform

47 igem The information has already about established other materials a functional other platform than for all the Bio-bricks participators are still to share missing Bio-bricks Bacteria strains Antibodies Cell strains Vectors Enzymes

48 Problems We May Have PROBLEM 1 New igemers may have trouble finding experiment materials 2 PROBLEM The reproducibility of previous igem projects may not be robust.

49

50 1 What s ishare?

51 Information-Sharing Platform ishare as we pictured is the first step to an all-round resource sharing platform. We suggest igem teams put information of materials used in their project on ishare and label those they are willing to share. In this way, future teams could have more resources for similar work and may come up with more improvements.

52 What information you can Get and Share on ishare Bacteria strains Antibodies Cell strains Vectors Enzymes

53 PLASMID All the information are as follows LacI Er R P laci Ap R P grax pht01 oriec ORF Name of Material Function Source of this Material Other Sources for Additional Information Can other teams directly get this material from you pht01 An expression vector we used in Bacillus amyloliquefaciens LL3 Mo Bi Tec(molecular biotechnology) NCBI YES

54 2 Survey on ishare

55 Survey on ishare Have you met any difficulties in finding experimental material related to your project? 19.32% 36.36% 3.41% 40.91% Yes, we have and our project was greatly delayed Yes, we have but it doesn't bother us No Others How difficult do you think it would be to repeat or improve previous igem projects? 56.82% 3.41% 34.09% 5.68% Difficult Easy It's hard to say Others Over 75% of them had difficulty finding experiment materials related to their projects. Only 6% of them think it s easy to repeat or improve previous igem projects.

56 Survey on ishare Would a Would resource you sharing like to platform be part of help ishare? in improving previous igem project? 15.91% 2.27% Yes, it will 0.00% 17.05% Yes, we'd No, love it won't to No, we won't It's hard to say 80.68% 84.09% Others Around 80% of the teams agreed that a resource sharing platform like ishare would help on solving the problems above. Up to 84% of the teams would like to be part of ishare

57 3 ishare Now and Future

58 HZAU-China LZU-China TJU NAU SCU-China Until now, 65 teams have shown the will to be part of BIT-China ishare. More and more teams are joining together SYSU-China FAFU-iGEM OUC-China Peking

59 ishare Now

60 ishare Future PC Software Mobile phone App

61 Welcome to ishare to share your information!!

62 Acknowledgement:

63 Cytotoxicity of γ-pgas/γ-pga hydrogels

64 Cytotoxicity of PINPAM & PINPAM/γ-PGA hydrogels Cytotoxicity of PINPAM Cytotoxicity of PINPAM /γ-pga hydrogels

65 Characteristics of γ-pgas/γ-pga hydrogels (C) Accumulated scavenging effect on superoxide Radicals of hydrogels for 12 h (D) Activity of released SOD

66 Wound healing effect Percentages of wound closure after distinct time intervals.

67 Wound healing effect

68 γ-pga s role in PNIPAM/γ-PGA hydrogels Contact angle of hydrogels with different γ-pga concentration. (A) PNIPAM/γ-PGA 50/50 (B) PNIPAM/γ-PGA 75/25 (C) PNIPAM/γ-PGA 100/0

69 Mathematical modeling Bacteria scale Product Substrate Bacteria scale during fermentation